PMN Adhesion Research Articles

Shiga Toxin 1 Induces on Lipopolysaccharide-Treated Astrocytes the Release of Tumor Necrosis Factor-alpha that Alter Brain-Like Endothelium Integrity

The hemolytic uremic syndrome (HUS) is characterized by hemolytic anemia, thrombocytopenia and renal dysfunction. The typical form of HUS is generally associated with infections by Gram-negative Shiga toxin (Stx)-producing Escherichia coli (STEC). Endothelial dysfunction induced by Stx is central, but bacterial lipopolysaccharide (LPS) and neutrophils (PMN) contribute to the pathophysiology. Although renal failure is characteristic of this syndrome, neurological complications occur in severe cases and is usually associated with death. Impaired blood-brain barrier (BBB) is associated with damage to cerebral endothelial cells (ECs) that comprise the BBB. Astrocytes (ASTs) are inflammatory cells in the brain and determine the BBB function. ASTs are in close proximity to ECs, hence the study of the effects of Stx1 and LPS on ASTs, and the influence of their response on ECs is essential. We have previously demonstrated that Stx1 and LPS induced activation of rat ASTs and the release of inflammatory factors such as TNF-α, nitric oxide and chemokines. Here, we demonstrate that rat ASTs-derived factors alter permeability of ECs with brain properties (HUVECd); suggesting that functional properties of BBB could also be affected. Additionally, these factors activate HUVECd and render them into a proagregant state promoting PMN and platelets adhesion. Moreover, these effects were dependent on ASTs secreted-TNF-α. Stx1 and LPS-induced ASTs response could influence brain ECs integrity and BBB function once Stx and factors associated to the STEC infection reach the brain parenchyma and therefore contribute to the development of the neuropathology observed in HUS.

Spreading of human neutrophils on an ICAM-1-immobilized substrate under shear flow

Neutrophil (PMN) spreading on endothelium, mediated by the interactions between surface-bound β2 integrin and intercellular adhesion molecule-1 (ICAM-1) in the inflammatory cascade, is crucial for PMN post-adhesion and trans-migration in blood flow. The underlying mechanisms by which shear flow regulates PMN spreading dynamics are not well understood. Here, a parallel-plate flow chamber assay was applied to quantify the time course of PMN adhesion and spreading on an ICAM-1-immobilized substrate. Two types of shear flow, steady flows at shear stresses of 0.2, 0.5, and 1 dyne/cm2 and stepwise flows at 0, 1, and 10 dyne/cm2, were used to elucidate the impact of shear flow on cell adhesion and spreading. The number of adhered PMNs, the fraction of spreading PMNs and the projected area of spread PMNs were determined and were found to correlate with the distribution of surface-bound β2 integrin subunit (CD11a, CD11b, or CD18). The results indicate that PMN spreading on an ICAM-1 substrate is bi-directionally regulated under shear flow. CD11a, CD11b and CD18 subunits of β2 integrin contribute distinctly to PMN spreading on ICAM-1 substrates. This work provides new insights into understanding PMN spreading on the endothelium, mediated by β2 integrin and ICAM-1 under shear flow.

Bacterial DNA Induces Pulmonary Damage Via TLR-9 Through Cross-talk With Neutrophils

Bacterial DNA (bDNA) contains hypomethylated "CpG" repeats that can be recognized by Toll-like receptor 9 (TLR-9) as a pathogen-associated molecular pattern. The ability of bDNA to initiate lung injury via TLR-9 has been inferred on the basis of studies using artificial CpG DNA. But the role of authentic bDNA in lung injury is still unknown. Moreover, the mechanisms by which CpG DNA species can lead to pulmonary injury are unknown, although neutrophils (PMNs) are thought to play a key role in the genesis of septic acute lung injury. We evaluated the effects of bDNA on PMN-endothelial cell (EC) interactions thought critical for initiation of acute lung injury. Using a biocapacitance system to monitor real-time changes in endothelial permeability, we demonstrate here that bDNA causes EC permeability in a dose-dependent manner uniquely in the presence of PMNs. These permeability changes are inhibited by chloroquine, suggesting TLR-9 dependency. When PMNs were preincubated with bDNA and applied to ECs or when bDNA was applied to ECs without PMNs, no permeability changes were detected. To study the underlying mechanisms, we evaluated the effects of bDNA on PMN-EC adherence. Bacterial DNA significantly increased PMN adherence to ECs in association with upregulated adhesion molecules in both cell types. Taken together, our results strongly support the conclusion that bDNA can initiate lung injury by stimulating PMN-EC adhesive interactions predisposing to endothelial permeability. Bacterial DNA stimulation of TLR-9 appears to promote enhanced gene expression of adhesion molecules in both cell types. This leads to PMN-EC cross-talk, which is required for injury to occur.

The Major Phagocyte Peroxidase Product HOSCN Stimulates PMN and Eosinophil Adhesion to Endothelium by p38 MAPK-Dependent Enhancement of CD11b/18-ICAM-1 Binding,

Abstract 3205Hypothiocyanous acid (HOSCN) is the predominant physiologic product of eosinophil peroxidase (EPO) and a major (50%) product of myeloperoxidase (MPO), a product of H2O2 oxidation of the pseudohalide substrate thiocyanate (SCN-). We have previously shown that this sulfhydryl-specific, cell-permeant oxidant, unlike HOCl, can potently induce expression of several NF-kB regulated genes, including the adhesion molecules ICAM-1 and VCAM-1, in human umbilical vein endothelial cells (HUVEC) through a transcriptional mechanism reflecting NF-κB activation. We here tested the hypothesis that HOSCN generated within activated PMN and eosinophils (EO) promotes their adhesion to ICAM-1 and VCAM-1 by enhancing the avidity for their leukocyte integrin counterligands, CD11b/18 (Mac-1) and CD49d/29 (VLA-4), respectively. We isolated human PMN and EO and tested the influence of reagent HOSCN upon their adhesion to purified human ICAM-1 and VCAM-1 immobilized in the bottom of bovine serum albumin (BSA)-blocked of tissue culture plate wells using a method based on crystal violet staining of nuclei and spectrophotometric assay of acetic acid extracts of adherent cells. 3–150 μM HOSCN enhanced adhesion to ICAM-1 of EO 10–18-fold over baseline binding and PMN 2–3-fold. This increase was inhibited >90% by both CBRM1/5, a blocking antibody against CD11b/18 and M1/70, which blocks ICAM-1, showing the specificity of this interaction. Similar concentrations of H2O2 did not significantly influence EO and PMN adhesion. HOSCN did not affect PMN or EO binding to immobilized VCAM-1. Pretreatment of EO with 33–150 μM HOSCN for 15 min prior to washing out HOSCN and layering them over TNF-α-activated HUVEC monolayers for 30 min stimulated a 5–15-fold increase in both EO and PMN adhesion that was > 75% inhibited by blocking antibodies against ICAM-1 and VCAM-1 (EO only) as well as CD11b. Assessing the potential mechanism behind HOSCN enhancing of CD11b/18-dependent EO and PMN adhesion, flow cytometry experiments showed HOSCN did not induce total cell surface expression of EO CD11b/18, but confocal microscopy showed prominent clustering of CD11b 20 min after exposure. Hypothesizing that the “stress” kinase p38 MAPK plays a role in HOSCN promotion of EO adhesion, we probed western blots whole cell lysates of EO exposed 1–10 min to 75 μM HOSCN for activated phospho-p38 MAPK and found a pronounced (10x baseline) but evanescent (1–2 min long) increase starting at 2 min. 1 μM SB203580, a potent and specific p38 MAPK inhibitor, blocked by >80% EO and PMN HOSCN stimulation of adhesion to both immobilized ICAM-1 and activated HUVEC. Collectively, these data show that HOSCN is a uniquely effective phagocyte oxidant inducer of both EO and PMN binding to ICAM-1 by a mechanism depending upon activation of p38 MAPK and enhancement of the CD11b/18-ICAM-1 interaction. This, in turn, suggests that endogenous MPO- and EPO-generated HOSCN may play a hitherto unsuspected physiologic autocrine amplification role in the regulation of adhesion and, hence, inflammation. In view of this, p38 MAPK inhibitors may have promise as therapeutic inhibitors in pathologic human inflammatory states, especially allergic diseases involving EOs. Disclosures:No relevant conflicts of interest to declare.

The nondepolarizing, normokalemic cardioplegia formulation adenosine-lidocaine (adenocaine) exerts anti-neutrophil effects by synergistic actions of its components

A new strategy of normothermic cardioplegia based on the combination of adenosine and lidocaine (adenocaine; Hibernation Therapeutics Global Ltd, Kilquade, Ireland) achieves nondepolarized arrest at normokalemia. Both adenosine and lidocaine independently inhibit neutrophil (polymorphonuclear neutrophil; PMN) activity. However, whether adenocaine exerts greater anti-inflammatory effects is not known. We tested the hypothesis that adenocaine synergistically attenuates PMN functions. Superoxide anion (O(2)(-)) generation: Isolated porcine PMNs were primed with cytochalasin B (5 μg/mL) and activated by N-formylmethionyl-leucyl-phenylalanine (100 nM). O(2)(-) release was quantified using lucigenin-enhanced chemiluminescence. Data were expressed as percent of stimulated control. Both adenosine and lidocaine alone inhibited O(2)(-) production in a dose-dependent manner (adenosine reduced to 67% ± 8.4% and 21% ± 2.2% of maximal stimulation at 0.1 and 10 μmol/L, respectively, lidocaine reduced to 57.9% ± 18.6% and 28% ± 5% at 10 and 100 μmol/L, respectively). Adenocaine further reduced O(2)(-) generation in a synergistic manner. In addition, adenosine alone (0.1-10 μmol/L) inhibited O(2)(-) generation in primed but not activated PMNs, whereas lidocaine alone (1-100 μmol/L) inhibited O(2)(-) release in both primed and activated PMNs. Adenocaine further reduced O(2)(-) generation because of inhibition of both priming and activation stages. Both adenosine and lidocaine alone and adenocaine comparably inhibited platelet activating factor-induced CD11 b/c surface expression on PMNs (flow cytometry), but adenocaine further suppressed both CD18 expression (to 47.4% ± 9.7%) and PMN adherence (to 47.2% ± 4.3%) compared with adenosine and lidocaine alone. Transmigration of calcein-acetyoxymethyl-labeled PMNs through transwells seeded with cultured coronary artery endothelial cells was reduced comparably by adenosine (to 80.1% ± 6.7%) and adenocaine (67.3% ± 9.6%). Adenocaine suppresses multiple PMN functions including O(2)(-) generation, adhesion molecule expression, PMN adherence, and transmigration. In addition to inducing nondepolarized arrest, adenocaine cardioplegia may exert cardioprotection by inhibiting PMN-mediated inflammatory responses.

Effect of intramammary infusion of rbGM-CSF on SCC and expression of polymorphonuclear neutrophil adhesion molecules in subclinical mastitis cows

The effect of rbGM-CSF intramammary infusion on the subclinical mastitis was evaluated by the somatic cell count (SCC) and expression of adhesion molecules (CD62L and CD11b) on the surface of neutrophils (PMN) in blood and milk. Fifteen cows diagnosed to have subclinical mastitis were used in this study. Seven cows showed a decrease in the SCC (decreased group), whereas 8 cows showed an increase in the SCC (increased group) 7 days after infusion of rbGM-CSF compared to pre infusion level. The percentage of CD62+ cells tended to be lower and CD11b+cells tended to be higher at 6 h on blood PMN in the decreased group of cows. Increased group of cows showed opposite tendencies. The mean fluorescent intensity of these adhesion molecules expressed on PMN in blood and milk was similar in both groups. These results suggested some association between expression of adhesion molecules and changes in SCC by rbGM-CSF. Responsiveness of PMN adhesion molecules to rbGM-CSF might determine the changes in SCC of the subclinical mastitic cows after infusion of rbGM-CSF.

Lung endothelial cell platelet-activating factor production and inflammatory cell adherence are increased in response to cigarette smoke component exposure.

An early event in the pathogenesis of emphysema is the development of inflammation associated with accumulation of polymorphonuclear leukocytes (PMN) in small airways, and inflammatory cell recruitment from the circulation involves migration across endothelial and epithelial cell barriers. Platelet-activating factor (PAF) promotes transendothelial migration in several vascular beds, and we postulated that increased PAF production in the airways of smokers might enhance inflammatory cell recruitment and exacerbate inflammation. To examine this possibility, we incubated human lung microvascular endothelial cells (HMVEC-L) with cigarette smoke extract (CSE) and found that CSE inhibits PAF-acetylhydrolase (PAF-AH) activity. This enhances HMVEC-L PAF production and PMN adherence, and adherence is blocked by PAF receptor antagonists (CV3988 or ginkgolide B). CSE also inhibited PAF-AH activity of lung endothelial cells isolated from wild-type (WT) and iPLA(2)β knockout mice, and with WT cells, CSE enhanced PAF production and RAW 264.7 cell adherence. In contrast, CSE did not affect PAF production or RAW 264.7 cell adherence to iPLA(2)β-null cells, suggesting that iPLA(2)β plays an important role in PAF production by lung endothelial cells. These findings suggest that inhibition of PAF-AH by components of cigarette smoke may initiate or exacerbate inflammatory lung disease by enhancing PAF production and promoting accumulation of inflammatory cells in small airways. In addition, iPLA(2)β is identified as a potential target for therapeutic interventions to reduce airway inflammation and the progression of chronic lung disease.

Intravenous immunoglobulin G (IVIG) inhibits IL-1- and TNF-α-dependent, but not chemotactic-factor-stimulated, neutrophil transendothelial migration

High-dose intravenous immunoglobulin (IVIG) has anti-inflammatory effects via incompletely understood mechanisms. By investigating whether IVIG might modulate neutrophil (PMN) recruitment, we observed that IVIG dose-dependently inhibited (by 30-50%) PMN transendothelial migration (TEM) across human umbilical vein endothelial cells (EC) stimulated with IL-1α, IL-1β, TNF-α or IL-1β+TNF-α. Inhibition required the presence of IVIG with the responding PMNs, was attributable to the F(ab)(2) portion and was unrelated to putative contaminants in IVIG. IVIG did not inhibit IL-1β- or TNF-α-induced increase of PMN adhesion to EC, nor did it affect C5a- or IL-8-induced PMN TEM across unstimulated EC. Effects of IVIG and F(ab)(2) fragments were not associated with PMN activation, assessed by CD62L shedding, CD11b upregulation or PMN shape. Thus, IVIG selectively inhibits PMN TEM across inflammatory-cytokine-stimulated - but not unstimulated - EC, perhaps contributing to therapeutic benefit in chronic inflammation with minimal impact on chemotactic-factor-induced PMN recruitment during acute infection.

Bradykinin and des-Arg10-kallidin enhance the adhesion of polymorphonuclear leukocytes to extracellular matrix proteins and endothelial cells

Bradykinin-related peptides (kinins) are well known to contribute to leukocyte recruitment to inflammatory foci; however, a role of these universal pro-inflammatory mediators in the first step of this process, i.e. the leukocyte adhesion to endothelial cells, is not well understood. In this work we found that bradykinin and des-Arg10-kallidin enhance the adhesion of polymorphonuclear bloods cells (PMN) to fibrinogen and fibronectin. Also, the PMN adherence to endothelial cells of HMEC-1 line strongly increased after stimulation by kinins, particularly des-Arg10-kallidin, or when PMN were co-stimulated with bradykinin and interleukin-1β. These effects were attenuated after PMN treatment with a specific inhibitor of carboxypeptidases, which convert kinins to their des-Arg metabolites. The kinin peptides were also able to change the Mac-1 integrin expression on the PMN surface. These results suggest a regulatory effect of kinins on leukocyte adhesion to endothelial wall, providing new aspects of the leukocyte infiltration into inflamed tissues.

Loss of p120 catenin upregulates transcription of pro-inflammatory adhesion molecules in human endothelial cells

P120 catenin (p120ctn) is an adherens junction protein recognized to regulate barrier function, but emerging evidence indicates that p120ctn may also exert control on other cellular functions such as transcriptional suppression of genes. We investigated the hypothesis that loss of p120ctn in human endothelial cells activates transcription of pro-inflammatory adhesion molecules. For study, siRNA targeted to p120ctn was transfected into brain microvascular (HBMECs) or pulmonary artery endothelial cells (HPAECs) for 24–120 h, which depleted 50–80% of endogenous p120ctn. This loss of p120ctn resulted in increased promoter reporter activity of transcription factors, NFκB, AP-1, and Kaiso, as well as of target genes, MMP-1 and ICAM-1. Real-time RT-PCR analysis indicated that the mRNA for ICAM-1, VCAM-1, and E- and P-selectins were all upregulated during the period of 24–120 h of p120ctn depletion, although the time-course and extent of the expression profiles differed. The upregulated mRNA of adhesion molecules corresponded with increased PMN adhesion to the EC surface and elevated ICAM-1 protein expression. We further explored the role of ERK1/2 as a potential signaling mechanism responsible for regulation of transcriptional activities by p120ctn. Results indicated that loss of p120ctn increased phosphorylated ERK1/2, and a MEK1 inhibitor (PD98059) prevented NFκB nuclear translocation. This implicates ERK1/2 in signaling the NFκB activation induced by p120ctn loss. The findings provide strong evidence that deficiency in p120ctn expression in endothelial cells is a potent stimulus for transcriptional upregulation of multiple adhesion molecules. We conclude that p120ctn functions to suppress transcription, which is an important and novel regulation in vascular endothelium.

GDF-15 is an inhibitor of leukocyte integrin activation required for survival after myocardial infarction in mice

Inflammatory cell recruitment after myocardial infarction needs to be tightly controlled to permit infarct healing while avoiding fatal complications such as cardiac rupture. Growth differentiation factor-15 (GDF-15), a transforming growth factor-β (TGF-β)-related cytokine, is induced in the infarcted heart of mice and humans. We show that coronary artery ligation in Gdf15-deficient mice led to enhanced recruitment of polymorphonuclear leukocytes (PMNs) into the infarcted myocardium and an increased incidence of cardiac rupture. Conversely, infusion of recombinant GDF-15 repressed PMN recruitment after myocardial infarction. In vitro, GDF-15 inhibited PMN adhesion, arrest under flow and transendothelial migration. Mechanistically, GDF-15 counteracted chemokine-triggered conformational activation and clustering of β(2) integrins on PMNs by activating the small GTPase Cdc42 and inhibiting activation of the small GTPase Rap1. Intravital microscopy in vivo in Gdf15-deficient mice showed that Gdf-15 is required to prevent excessive chemokine-activated leukocyte arrest on the endothelium. Genetic ablation of β(2) integrins in myeloid cells rescued the mortality of Gdf15-deficient mice after myocardial infarction. To our knowledge, GDF-15 is the first cytokine identified as an inhibitor of PMN recruitment by direct interference with chemokine signaling and integrin activation. Loss of this anti-inflammatory mechanism leads to fatal cardiac rupture after myocardial infarction.

Src Phosphorylation of Endothelial Cell Surface Intercellular Adhesion Molecule-1 Mediates Neutrophil Adhesion and Contributes to the Mechanism of Lung Inflammation

The goal of this study was to determine whether tumor necrosis factor α (TNFα)-induced Src activation and intercellular adhesion molecule-1 (ICAM-1) phosphorylation rapidly increase endothelial cell adhesivity and polymorphonuclear leukocyte (PMN) sequestration independently of de novo ICAM-1 synthesis. TNFα exposure of mouse lungs for 5 minutes produced a 3-fold increase in (125)I-anti-ICAM-1 monoclonal antibody (mAb) binding and (111)In oxine-labeled PMN sequestration, as well as Src activation, ICAM-1 Tyr518 phosphorylation, and phospho- Tyr518-ICAM-1 coimmunoprecipitation with actin. The response was absent in Nox2(-/-) lungs or following Src inhibition. In COS-7 cells transfected with wild-type (WT), phospho-defective (Tyr518Phe), or phospho-mimicking (Tyr518Asp) mouse ICAM-1 cDNA constructs, TNFα increased the B(max) of YN1/1.7.4 anti-ICAM-1 mAb binding to WT-ICAM-1 but not to Tyr518Phe-ICAM-1, indicating increased binding avidity secondary to ICAM-1 phosphorylation. This effect was mimicked by expression of the Tyr518Asp-ICAM-1 mutant. TNFα also increased the staining intensity and cell surface clustering of YN1/1.7.4 mAb-labeled WT-ICAM-1 that colocalized with F-actin, which was not observed with Tyr518Phe-ICAM-1 but was recapitulated with Tyr518Asp-ICAM-1. Finally, overexpression of ICAM-1 in mouse lungs significantly increased lipopolysaccharide-induced transvascular albumin leakage and bronchoalveolar lavage PMN counts at 2 and 24 hours after lipopolysaccharide inhalation compared with lungs expressing the Tyr518Phe ICAM-1 mutant. Src-dependent phosphorylation of endothelial cell ICAM-1 Tyr518 induces PMN adhesion by promoting ICAM-1 clustering, which we propose mediates rapid-phase lung vascular accumulation of PMNs during inflammation.

Attenuation of human neutrophil migration and function by uropathogenic bacteria

The establishment of bacterial infections at mucosal epithelial surfaces is determined by the balance of virulence attributes of the pathogen with the activity of innate host defenses. Polymorphonuclear leukocytes (PMN) are key responders in many bacterial infections, but the mechanisms by which pathogens subvert these early responses to establish infection are largely undefined. Here, we model early interactions between human PMN and the primary cause of urinary tract infections, namely uropathogenic Escherichia coli (UPEC). Our objective was to define virulence phenotypes of uropathogens that permit evasion of PMN activity. We show that UPEC strains, as compared with laboratory and commensal E. coli, resist phagocytic killing and dampen the production of antimicrobial reactive oxygen species by PMN. Analysis of the transcriptional responses of PMN to E. coli strains revealed that UPEC exposure downregulates the expression of PMN genes that direct pro-inflammatory signaling and PMN chemotaxis, adhesion, and migration. Consistent with these data, UPEC attenuated transepithelial neutrophil recruitment in an in vitro model of acute infection and in a murine model of bacterial cystitis. We propose that these UPEC strategies are important in the establishment of epithelial infection, and that the findings are germane to bacterial infections at other epithelial surfaces.

Cytohesin-1 regulates fMLF-mediated activation and functions of the β2 integrin Mac-1 in human neutrophils

The nucleotide exchange factor cytohesin-1 was previously reported to interact with the cytoplasmic domains of the integrin β-chain common to all β(2) integrins such as LFA-1 and Mac-1. We show here that cytohesin-1, which contributes to fMLF-induced functional responses in PMNs through activation of Arf6, restrains the activation of the β(2) integrin Mac-1 (αMβ(2)) in PMNs or dcAMP-differentiated PLB-985 cells. We found that the cytohesin-1 inhibitor SecinH3 or siRNA increased cell adhesion to immobilized fibrinogen and fMLF-mediated conformational changes of Mac-1, monitored using mAb CBRM1/5, specific for the activation epitope of the αM subunit. In contrast, PLB-985 cells overexpressing cytohesin-1 showed little adhesion to fibrinogen. The use of SecinH3 and siRNA also revealed that interference with cytohesin-1 signaling also enhanced phagocytosis of zymosan particles and chemotaxis toward fMLF in transwell migration assays. These increments of phagocytosis and chemotaxis in cells treated with SecinH3 and cytohesin-1 siRNA were reversed by a blocking mAb to the integrin-αM subunit. We provide evidence for increased polymerized cortical actin in cells treated with SecinH3 and that altered signaling through cytohesin-1 increased cell surface expression of FPRL-1 and impairs the late calcium mobilization response elicited by fMLF. The data provide evidence that stimulation with fMLF initiates a signaling cascade that restrains Mac-1 activation in PMNs. Such crosstalk between FPRL-1 and Mac-1 involves cytohesin-1. We suggest that cytohesin-1 may coordinate activation of the β(2) integrins to regulate PMN adhesion, phagocytosis, and chemotaxis.

Urothelial Cell Platelet-activating Factor Production Mediated by Calcium-independent Phospholipase A 2γ

To determine the effect of phospholipase A(2) (PLA(2)) inhibitors on urothelial cell platelet-activating factor (PAF) production in response to tryptase stimulation. Urothelial cells isolated from normal human ureters were immortalized with the human papillomavirus type 16E6E7 cell line (TEU-2 cells). PLA(2) activity in TEU-2 cells was measured using (16:0, [(3)H]18:1) plasmenylcholine and phosphatidylcholine substrates in the presence and absence of calcium. [(3)H]PAF production was measured in TEU-2 cells prelabeled with [(3)H] acetic acid. PAF-acetylhydrolase activity was measured by determining the amount of [(3)H] acetate hydrolyzed from [(3)H]PAF incubated with TEU-2 cellular protein. Adherence of human polymorphonuclear leukocyte (PMN) to TEU-2 cells was assessed by measuring myeloperoxidase activity in adherent PMNs after incubation with TEU-2 cells. Most PLA(2) activity measured in TEU-2 cells was determined to be membrane-associated, calcium-independent PLA(2) and selective for plasmenylcholine substrate. Stimulation of TEU-2 cells with tryptase results in increased production of PAF and increased PMN adherence that were inhibited completely by pretreatment with the membrane-associated, calcium-independent PLA(2)γ-selective inhibitor (R)-bromoenol lactone. Pretreatment with the cytosolic PLA(2) inhibitor methyl arachidonyl fluorophosphonate resulted in potentiation of tryptase-stimulated PAF production and PMN adherence to TEU-2 cells that is a result of PAF-acetylhydrolase inhibition. Tryptase stimulation of TEU-2 cells results in activation of membrane-associated, calcium-independent PLA(2)γ, leading to an increase in PAF production and increased PMN adherence. Inhibition of TEU-2 cell PAF-acetylhydrolase activity with methyl arachidonyl fluorophosphonate potentiated tryptase-stimulated PAF production and PMN adherence.

α-Enolase From Injured Patients and Stored Packed Red Blood Cells Activates Pulmonary Endothelium and Serves as the First Event In a Two-Event Model of Neutrophil Cytotoxicity

AbstractAbstract 3355A significant number of injured patients with intermediate injury severity scores (15<ISS<30) develop multiple organ failure (MOF), which clinically begins with acute lung injury (ALI). Transfusion of >6 units of stored PRBCs (≥28 days) is associated with the development of ALI/MOF on day 3 post injury (Am J Surg 178:502-4, 1999). The pro-inflammatory mediators, e.g. cytokines, responsible for MOF/ALI in the injured have remained elusive; therefore, we hypothesize that “moonlighting” proteins, which have defined intracellular function when released in the circulation, activate innate immunity and are etiologic in the development of ALI/MOF post-injury. Methods:Proteomics on the field blood (plasma) of 3 patients with blunt trauma who later developed ALI/MOF and the plasma from 3 units of packed red blood cells (PRBCs) on day 1 and day 42 were completed using 2-dimensional gel electrophoresis/mass spectroscopy (MALDI/TOF) with computer analyses of the resultant peptides. The proteins from whole cell lysates from Human pulmonary microvascular endothelial cells (HMVECs) were separted by SDS-PAGE, transferred to nitrocellulse and immunoblotted with antibodies to protease activated receptor-1 (PAR-1) and PAR-2. HMVECs were also incubated for 6 hours and 1) ICAM-1 was measured by flow cytometry, 2) isolated neutrophils (PMNs) were added allowed to settle and in selected wells PMN adherence to these activated HMVECs was measured by myeloperoxidase content in the lysate, or 3) after the PMNs settled, lysophosphatidylcholines (lyso-PCs) [4.5μM], lipids from stored platelets implicated in TRALI, were added and the number of viable HMVECs/mm2 were counted by microscopy. Results:HMVECs display immunoreactivity for both PAR-1 and PAR-2. Of the 243 proteins identified in the injured patients and the stored vs. fresh PRBCs, α-enolase increased by 10.8-fold and 4.4-fold respectively (p<.05 & p<.005). Both thrombin and α-enolase induced ICAM-1 expression in HMVECs (Table 1) which was inhibited (60±8%) by pre-treatment with the anti-protease leupeptin. α-Enolase also induced significant PMN adhesion vs. media control: Media: 3.1±1.5; α-enolase (50 μg/ml): 14.4±4.7; LPS: 35.5±0.7*. The α-enolase-activated HMVECs vs. buffer-treated lyso-PCs induced significant PMN-mediated cytotoxicity (Table 2). We conclude that α-enolase from the injured and stored but not fresh PRBCs causes pro-inflammatory activation of HMVECs resulting in PMN adherence and PMN cytotoxicity in a two-event in vitro model through activation of PARs receptors. Moonlighting proteins like the glycolytic lyase α-enolase may have unexpected pro-inflammatory activity, which predispose the injured patient to increased morbidity and mortality. Disclosures:No relevant conflicts of interest to declare.

The Major Phagocyte Peroxidase-Derived Oxidant HOSCN Stimulates Gene-Specific p38 MAPK- Dependent, NF-κb-Mediated Activation of Tissue Factor, VCAM-1 and ICAM-1 Expression In Endothelium.

AbstractAbstract 1481Thiocyanate (SCN-) is, unexpectedly, the principal physiologic substrate for eosinophil peroxidase (EPO) and a major (i.e., accounting for 50% of H2O2 consumed) substrate for myeloperoxidase (MPO). The product of these reactions is HOSCN, a weak, exclusively sulfhydryl-reactive oxidant that we have previously shown to be a uniquely potent (up to 100-fold) oxidant transcriptional inducer of human umbilical vein endothelial cell (HUVEC) tissue factor (TF), ICAM-1 and VCAM-1 expression via a mechanism dependent upon NF-κB p65/p50 activation. We hypothesized that oxidative activation of p38 MAPK is a necessary and proximal step in the activation of NF-κB p65/p50 at the TF, VCAM-1 and ICAM-1 promoters. To test this we utilized the p38 MAPK-specific pyridinyl imidazole inhibitor SB 203580 (SB). In HUVEC monolayers exposed 4h to 150 μM HOSCN in M199 medium containing 10% FCS, SB strongly inhibited (>90%, ED50 300 nM) TF activity, decreased VCAM-1 expression assessed by western blot by > 50% but had no discernible effect upon ICAM-1 expression. qRT-PCR analysis confirmed that the effects of SB on these three molecules was transcriptionally mediated. Immunoprecipitation of HOSCN-treated HUVEC whole cell extracts with a polyclonal anti-phospho-p38 MAPK reagent showed rapid (within 1 min) phosphorylation of p38 MAPK that lasted >30 min with consequent kinase activity documented by detection of the downstream target product phospho HSP27. Confocal immunofluorescence confirmed the rapid and durable induction of phospho p38 MAPK from undetectable to abundant, primarily cytoplasmic, but also some nuclear localization. We further hypothesized that the disparate effects of SB on these three molecules reflects differences in the sequences of their NF-κB-binding sites that affect their ability to bind the p65 NF-kB subunit. Chromatin immunoprecipitation (ChIP) analysis of HUVEC treated for 3h with HOSCN using an anti-p65 antibody revealed that SB inhibited by 75% the 10-fold increase in binding of p65 to the authentic endogenous VCAM-1 and NF-κB binding loci but had no discernible effect on p65 binding to the ICAM-1 locus. An identical pattern was seen in HUVEC exposed 30 min to the prototypical inflammatory cytokine TNF. To test the physiologic significance of these findings we assessed the effect of SB upon PMN and eosinophil binding to HOSCN-exposed HUVEC monolayers treated as described above. SB pretreatment of HUVEC blocked by > 80% the 3–8-fold increase in binding of PMN and eosinophils that occurs in HOSCN-exposed monolayers. Blocking antibodies to ICAM-1 and VCAM-1 demonstrate that both of the adhesion molecules contribute to HOSCN-induced PMN and eosinophil adhesion. We conclude that HOSCN generated by PMN and eosinophils attached or subjacent to vascular endothelium has the capacity to rapidly activate endothelial p38 MAPK-dependent activation of p65 binding to the TF and VCAM-1, but not ICAM-1, NF-κB binding loci; and these differences manifest in physiologically relevant transcriptional regulation of protein expression. Therefore, p38 MAPK inhibitors may have anti-thrombotic and anti-inflammatory potential, the latter particularly in states, such as allergic inflammation, that are most dependent on VCAM-1. Disclosures:No relevant conflicts of interest to declare.

Expression of P-Selectin and Platelet-Neutrophil (PMN) Interactions In Untreated and Mirasol® PRT Treated Platelet Concentrates During Routine Storage.

AbstractAbstract 1123 Introduction:Platelet concentrates develop biologically active compounds during storage which may play a role in adverse events of transfusion. Although many studies have focused on release of soluble pro-inflammatory compounds, changes in cells such as platelets may contribute to the pro-inflammatory effects of transfusion products. We evaluated untreated and Mirasol Pathogen Reduction Technology (PRT) treated platelet concentrates for expression of P-selectin and interactions between platelets and PMNs. Methods:Four, double apheresis platelet products were collected from healthy volunteers with TRIMA system under an IRB approved protocol. One product of each pair was PRT treated with Mirasol PRT and both were stored under standard conditions. At various times, aliquots were sterilely removed from products. Expression of P-selectin on platelets was defined with a specific antibody. Neutrophils, isolated from ABO type specific peripheral blood of volunteers, were incubated with platelet concentrates for 3 min at 37° C, and labeled CD41 was added. PMNs were identified with flow cytometry by forward/side scatter and platelets by fluorescence and the % of PMNs associated with platelets determined. Inhibition of platelet/neutrophil interaction with antibodies to adhesion molecules was also examined. In parallel experiments, activation of neutrophils after incubation with platelets was determined by oxidation of dihydrorhodamine (DHR). Results:During storage, platelet expression of P-selectin increased in both untreated and Mirasol treated platelet concentrates (p<0.05) with greater expression on treated products (p<0.05). On day 0 of storage, PMNs associated with platelets was 9.7±1.1% (mean±SEM, n=4, untreated) and 12.1±1.3% (treated products), and on day 5 results were 16.8±4.3% and 27.1±4.3% respectively (significant, p<0.05). Platelet-PMN interactions were inhibited by ≥90% by antibodies to P-selectin or its PMN adhesion partner PSGL-1, but not Mac1, LFA-1, GPIb or ICAM-1. Platelet-PMN interactions were directly related to the stored platelets, not the effects of stored plasma. Activation of the PMN respiratory burst by oxidation of DHR was not shown in any product type over storage time. Conclusion:During storage, platelets exhibit increased P-selectin expression and the capability to adhere to PMNs. These effects were greater after Mirasol PRT treatment. The cell association depended on the interaction between P-selectin (platelets) and PSGL-1 (PMNs). These changes occurring during storage of platelet concentrates may explain platelet recovery patterns in patients requiring repeated transfusions with untreated and PRT treated products and could play a potential role in adverse events for either product type. Disclosures:Goodrich:CaridianBCT Biotechnologies: Employment, Patents & Royalties, Research Funding. Marschner:CaridianBCT Biotechnologies: Employment, Research Funding.

Neutrophils Clearance by Endothelial Cells Regulates Homeostasis and Coagulation.

AbstractAbstract 3782Neutrophils, also called polymorphonuclear leukocytes (PMN's) have a half-life of only 6 hours in the blood. Inflammation can further shorten the circulating life-time. A large fraction of the bone marrow capacity is committed to ongoing production of these short-lived cells but the manner of their clearance from the circulation is less well understood. We have previously demonstrated that PMN's are cleared by liver macrophages. However, the details of PMN adhesion-induced PMN clearance in the liver are unknown. The aim of this study is to evaluate a pathway of PMNs clearance by endothelial cells, which are not ordinarily considered phagocytes. Lactadherin is a glycoprotein of milk fat globules and is also secreted by stimulated macrophages. Lactadherin binds phosphatidylserine on apoptotic cells via tandem lectin-homology domains with homology to factor VIII and binds αvβ3 and αvβ5 integrins on phagocytic cells via an RGD sequence in an epithelial growth factor domain. Lactadherin aids in engulfment of senescent lymphocytes by splenic macrophages and mediates an anti-inflammatory response. We utilized lactadherin as a probe to detect phosphatidylserine exposure on aging PMN's and evaluated the lactadherin-dependent engulfment of these PMN's by endothelial cells. Cultured human PMNs from healthy donors, with 95% purity, were 40% and 96% PS-exposure positive at 9 and 24 h, respectively. They displayed a parallel increase in procoagulant supporting, activity related to the PS exposure. Coculture of the aging PMNs and human umbilical vein endothelial cells resulted in phagocytosis of the PMN's, observed by confocal microscopy and electron microscopy. Exogenous lactadherin increased phagocytosis by 3–5 fold during 120 minutes of observation. An anti-lactadherin RGD antibody and an anti-lactadherin C2 domain antibody inhibited phagocytosis to approx 1/2 the background level suggesting that lactadherin secreted by PMN's or neutrophils contributes to the base level of phagocytosis. Clearance of the senescent neutrophils by endothelial cells decreased procoagulant activity >70% and blockade of neutrophil PS with lactadherin reduced procoagulant activity by > 90% indicating the potential role of neutrophil uptake in limiting prothrombotic activity. In a rat model of neutrophil homeostasis we injected low dose lipopolysaccharide (LPS) and gadolinium chloride intravenously to increase the number circulating PMN's and block clearance by Kupffer cells. This allowed observation of PMN adhesion and sequestration in the liver. The number of PMNs peaked at 9 h and decreased to the normal range at 24 h after blockade of Kupffer cells. Blocking the endothelial P-selectin significantly delayed PMN's removal in the liver. Injection of lactadherin promoted the PMNs accumlation and removal. The current results suggest that ECs contribute to maintaining the homeostasis of PMNs in the circulation and a possible role of lactadherin in the EC-mediated clearance. Our results also indicate that lactadherin-mediated clearance may limit procoagulant or prothrombotic activity of senescent PMN's. Disclosures:No relevant conflicts of interest to declare.

E0047 Alphalinonenic acid inhibits high glucosemediated endothelial neutrophil adhesion by decreasing adhesion molecule expression via PI3KAkt pathway

ObjectiveNeutrophil-endothelial adhesion is crucial to vascular injury, the major cause of diabetic vascular complications. We studied the mechanism of cardio-protective effect of Alpha-linolenic acid (ALA).MethodsHuman umbilical vein endothelial cells (HUVECs)...